Hisaya Tada

An aldose reductase inhibitor prevents glucose-induced increase in transforming growth factor-β and protein kinase C activity in cultured human mesangial cells

Summary We investigated the effect of inhibition of a polyol pathway on the glucose-induced increase in transforming growth factor-β (TGF-β) production and activity of protein kinase C (PKC) in cultured human mesangial cells (MCs). The exposure of MCs to 33 mmol/l glucose resulted in an increase in TGF-β production, measured by ELISA, compared with 5 mmol/l glucose. The glucose-induced increase in TGF-β was prevented by concomitant incubation with epalrestat, an aldose reductase inhibitor (ARI), in a dose-dependent manner at a concentration of more than 10–6 mol/l. Moreover, the glucose-induced enhancement of PKC activity in the membrane fraction of MCs was also abolished by epalrestat. The addition of epalrestat to MCs cultured with 5 mmol/l glucose showed no demonstrable effects on TGF-β production and PKC activity. These results provide direct evidence for linkages between derangements in polyol pathway and glucose-induced overproduction of TGF-β and enhancement of PKC activity in MCs. Accordingly, the effect of an ARI on these metabolic abnormalities in MCs may justify its clinical application for treatment of diabetic nephropathy. [Diabetologia (1998) 41: 362–364]

Long-term effect of epalrestat, an aldose reductase inhibitor, on the development of incipient diabetic nephropathy in Type 2 diabetic patients.

The aim of the present study was to elucidate the long-term effect of epalrestat, an aldose reductase inhibitor (ARI), on renal function in patients with type 2 diabetes mellitus showing microalbuminuria. Patients were allocated to two groups (cases and controls) matched for age, BMI, and the extent of urinary albumin excretion (UAE). Thirty-five type 2 diabetic patients presenting microalbuminuria were included in this study: cases were treated with epalrestat (150 mg/day) for 5 years. No significant changes were found in blood pressure, HbA1c, and total cholesterol in either group during the observation period. In the control group, UAE increased significantly (P<.01) from 82+/-12 mg/g Cr at the baseline to 301+/-111 mg/g Cr at the end of the study, while UAE remained unchanged, 81+/-15 mg/g Cr at the baseline and 87+/-19 mg/g Cr at the end of the study, in the epalrestat-treated group. Reciprocal creatinine measured by an enzyme assay decreased significantly (P<.01) in both groups; however, the reduction rate in the epalrestat-treated group was significantly (P<.05) smaller than that in the control group. These results suggest the potential usefulness of ARIs in preventing the progression of incipient diabetic nephropathy in patients with type 2 diabetes mellitus.

The Fibronectin Production Is Increased by Thrombospondin via Activation of TGF-β in Cultured Human Mesangial Cells

Thrombospondin (TSP) is a multifunctional glycoprotein that is synthesized by a variety of cells including mesangial cells (MCs). To clarify the effect of TSP on the pathogenesis of diabetic nephropathy, we studied the effect of glucose concentrations on TSP synthesis in cultured human MCs. Thereafter, the effects of TSP on the activation of transforming growth factor beta (TGF-β) and fibronectin production were investigated in MCs. Incubating MCs with elevated glucose levels for 6 days resulted in an increase in TSP synthesis, measured by an enzyme-linked immunosorbent assay, both in culture media and cell layers. Treatment of MCs with TSP (final concentrations 1 and 5 µg/ml) for 24 h resulted in an increase (1.3- and 2.1-fold, respectively) in active TGF-β, which was determined with an enzyme-linked immunosorbent assay using TGF-β-soluble receptor type II, in the culture media without having any effect on the production of total TGF-β. Exposure of MCs to TSP caused enhancement of fibronectin production in both media and cell layers in a TSP dose-dependent manner with the maximum at a TSP concentration of 1 µg/ml. The TSP-induced increase in fibronectin production from MCs was completely prevented by concomitant treatment with 10 µg/ml anti-TGF-β neutralizing antibody. These results indicate that the TSP production is promoted by a high ambient glucose concentration in human MCs and that TSP, in turn, causes an increase in fibronectin production via activation of TGF-β.

Protective effect of d-α-tocopherol on the function of human mesangial cells exposed to high glucose concentrations

Altered functions of mesangial cells (MCs) induced by high glucose levels are thought to play an important role in the pathogenesis of diabetic nephropathy. We investigate whether D-alpha-tocopherol (Toc), an antioxidant, can prevent malfunction of cultured human MCs induced by high-glucose media. Incubating MCs with 33 mmol/L glucose caused increased lipid peroxide (LPO) levels, disturbed cell replication, enhanced cytotoxicity, enhanced activity of the diacylglycerol (DAG)-protein kinase C (PKC) pathway, and overproduction of fibronectin and eicosanoids (6-keto prostaglandin F1 alpha [PGF1 alpha] and thromboxane B2 [TXB2]). The amount of LPO in MCs grown in 5 mmol/L glucose was reduced by the addition of Toc in a dose-dependent manner. Since the maximum effect of Toc on decreasing LPO was achieved at a concentration of 100 mumol/L, this dose was selected for the following experiments. Addition of Toc prevented increased LPO levels and [51Cr]-release from MCs induced by high-glucose media without affecting cell number. Toc decreased the total DAG level and PKC activity in membrane fractions in MCs cultured at both 5 and 33 mmol/L glucose. Furthermore, glucose-induced overproduction of fibronectin and eicosanoids from MCs was completely abolished by Toc. These results strongly suggest that Toc ameliorates glucose-induced malfunctions of MCs in vitro.

Role of protein kinase C-angiotensin II pathway for extracellular matrix production in cultured human mesangial cells exposed to high glucose levels.

The intrarenal renin-angiotensin system has been implicated in the pathogenesis of diabetic nephropathy. This study investigates the mechanisms for glucose-induced increase in angiotensin II (AII) production by human mesangial cells (MCs) in relation to protein kinase C (PKC). We also examine whether locally produced AII mediates extracellular matrix protein production in high-glucose conditions. Human MCs were cultured in 5 or 33 mmol/l glucose for 8 days, and were incubated with or without 5 mmol/l GFX, a PKC inhibitor, 0.1 micromol/l candesartan cilexetil (CC), a specific type 1 AII receptor antagonist, for another 24 h. In addition, MCs grown in 5 mmol/l glucose were incubated with 0.1 micromol/l phorbol-12,13-dibutyrate (PDBu) for 24 h. AII, TGF-beta1, fibronectin and type IV collagen in the culture media were measured by ELISA. The amount of AII secreted from MCs exposed to high-glucose levels was significantly greater (P<0.01) than that in normal glucose levels. The increase in AII production was completely prevented by GFX. The addition of PDBu mimicked the effect of glucose on AII production. The glucose-induced increases in the production of TGF-beta1, fibronectin and type IV collagen were partially, but significantly restored (P<0.01) by CC, while GFX totally abolished these effects of glucose. These results suggest that elevated glucose levels stimulate AII production via mechanisms dependent on glucose-induced PKC activation in human MCs, and that locally produced AII partly mediates the increase in mesangial matrix synthesis in high-glucose conditions.

High glucose levels enhance TGF-β1–thrombospondin-1 pathway in cultured human mesangial cells via mechanisms dependent on glucose-induced PKC activation

We have previously demonstrated that thrombospondin-1 (TSP-1) promotes activation of latent TGF-beta1 in cultured human mesangial cells (MCs) [Nephron 79 (1998) 38.]. This study was performed to clarify the relationship between protein kinase C (PKC) activity and activation of TGF-beta1 and TSP-1 production in cultured human MCs exposed to high glucose levels. MCs grown in 33 mmol/l glucose demonstrated a significant (P< .01) increase in activation of TGF-beta1 compared with those in 5 mmol/l glucose, which were evaluated by both an ELISA and a bioassay. High glucose-induced increase in active TGF-beta1 was completely inhibited by coincubation with 5 micromol/l GFX, a PKC inhibitor, and was mimicked by the addition of 0.1 micromol/l phorbol 12,13-dibutyrate (PDBu). On the other hand, increased TSP-1 production from MCs stimulated by 0.1 nmol/l recombinant TGF-beta1 and 0.1 micromol/l PDBu were significantly (P< .01) reduced by the addition of 10 nmol/l latency-associated peptide (LAP), a specific inhibitor of TGF-beta1 activity. The amount of TSP-1 secreted from MCs increased by a high ambient glucose. The glucose-induced increase in TSP-1 production was markedly attenuated by the treatment with GFX and LAP, while those agents did not affect TSP-1 production in low-glucose concentrations. Taken together, our results suggest that glucose-induced activation of TGF-beta1 is dependent on PKC activity, leading to a sequential increase in TSP-1 synthesis in cultured human MCs. Thus, we propose that high glucose conditions induce an increase in PKC-TGF-beta1 activity-TSP-1 pathway, and that glucose-induced increase in TSP-1 may synergistically facilitate TGF-beta1 activation in an autocrine manner in MCs.

A high concentration of glucose alters the production of tPA, uPA and PAI-1 antigens from human mesangial cells.

To elucidate a role of tPA, uPA and PAI-1 for the development of diabetic glomerulosclerosis, the effect of high glucose concentration on the production of both basal and thrombin-mediated tPA, uPA and PAI-1 antigens from human mesangial cells was investigated. The culture of mesangial cells in the presence of high glucose (33 mM) for 11 days resulted in an increase in the synthesis of tPA and uPA when compared with that in normal glucose concentration (5 mM). In contrast, the cells grown in high glucose produced less PAI-1 than those in normal glucose. Thrombin stimulated dose-dependently the production of tPA, uPA and PAI-1 from the cells grown in either 5 or 33 mM glucose. However, the magnitude of the increase in tPA, uPA and PAI-1 from the cells grown in high glucose was less than that in normal glucose. These results suggest that the plasmin activity in mesangial cells may increase under a high glucose condition, leading to increased proteolysis of mesangial matrix. In addition, either fibrinolysis or proteolysis mediated by thrombin may be altered by high glucose concentration. Therefore, it is postulated that the turnover of mesangial matrix may be increased in diabetic nephropathy.

Delapril versus manidipine in hypertensive therapy to halt the type-2-diabetes-mellitus-associated nephropathy

Thirty-nine hypertensive patients with type 2 diabetes mellitus were followed under long-term treatment (mean, 20.7 months) with manidipine hydrochloride, a Ca antagonist, or delapril hydrochloride, an ACE inhibitor, at nine institutions. Both the treatments showed similar antihypertensive effects, although slight but significantly larger decreases were observed in systolic and mean blood pressures at months 12 and 24 in the patients treated with manidipine (P < 0.02). The urinary albumin excretion index (AEI) tended to increase throughout the study in both treatment groups, but no significant difference in AEI was observed between the two treatment groups at any time point. Overt albuminuria developed in four patients on manidipine but did not appear in any of the patients on delapril. The risk of progression to overt albuminuria was significantly different between manidipine and delapril groups (P = 0.011). No increase in serum creatinine (Cr) was observed with delapril. The average excretion indexes of tubular markers such as beta2-microglobulin, alpha1-microglobulin, and NAG tended to be higher in the patients on manidipine than in those on delapril. Taken in sum, these findings suggest that the ACE inhibitor delapril is more beneficial than the Ca antagonist manidipine in the treatment of diabetic renal diseases via mechanisms other than the blood pressure regulation, partly through their different effects on tubular function. In conclusion, delapril was significantly more effective than manidipine in inhibiting progression to overt albuminuria in hypertensive type 2 diabetes mellitus patients.

Relationship between urinary excretion of fibronectin degradation products and proteinuria in diabetic patients, and their suppression after continuous subcutaneous heparin infusion

To explore the possibility that the excretion of urinary fibronectin degradation products (U-FnDP) can be an indicator of the progression of diabetic nephropathy, U-FnDP and urinary protein(U-P) were determined in 64 diabetic patients and 11 healthy volunteers. Moreover, to determine whether continuous subcutaneous heparin infusion (CSHI) reduces elevated U-FnDP and U-P in diabetic patients with persistent proteinuria, heparin sodium was administered as a bolus subcutaneous injection of 5000 IU, followed by subcutaneous infusion of 250 IU/kg per 24 h heparin sodium for 7 days. U-FnDP excretion rate elevated proportionally to the degree of U-P. CSHI reduced significantly elevated U-FnDP from 172.68 +/- 15.79 to 100.04 +/- 14.93 micrograms/24 h (P < 0.01) and U-P from 1.76 +/- 0.13 to 1.20 +/- 0.12 g/24 h (P < 0.01). No significant changes in blood pressure and diurnal mean plasma glucose levels were found. APTT was prolonged with a decrease of AT-III activity during the treatment. These findings suggest that U-FnDP can be one of the indicators which reflects the degree of progression of diabetic nephropathy, and that CSHI may be useful for the normalization of elevated U-FnDP and reduction in U-P in diabetic nephropathy.

Increased intraglomerular thrombin formation in diabetic microangiopathy

Estimations of soluble fibrin monomer complexes (SFMC) in plasma are a convenient index of thrombin activation. Renal venous-arterial differences in plasma SFMC concentrations were determined in 16 randomly chosen diabetic patients by sampling directly and simultaneously from the renal artery and vein according to the method of Seldinger. In all subjects, SFMC concentrations were higher in the renal vein than in the renal artery, indicating that the kidney is an important source of SFMC. Venous-arterial differences were markedly elevated in patients with severe renal and retinal microangiopathy coupled with hypertension. The hypothesis is advanced that elevated plasma SFMC levels lead to abnormal fibrin deposits in lesioned glomeruli and retinal vessels. It is postulated that plasma SFMC may be a useful parameter for the assessment of diabetic vascular complications.